Decontamination of biological fluids using diphenylpyrilium compounds

ABSTRACT

The present invention affords means for decontaminating biological fluids such as blood and blood components. The method involves contacting a biological fluid with a diphenylpyrilium compound, and irradiating the mixture with red light. The method is a potent and effective means for eliminating or diminishing active pathogens such as viruses, bacteria, and parasites, without causing substantial hemolysis or otherwise degrading the storage stability of the decontaminated biological fluid.

REFERENCE TO PRIOR APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/475,493, filed Jun. 4, 2003.

GOVERNMENT LICENSE RIGHTS

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant no. HL66779 awarded by the NIH National Heart Lung and Blood Institute.

BACKGROUND OF THE INVENTION

Over the past two to three decades, numerous donor screening andinfectious disease testing methods have been implemented to reduce thetransmission of human immunodeficiency virus (HW), hepatitis C virus(HCV), hepatitis B virus (OBV), and human T-cell lymphotropic viruses 1and 2 (HTLV-1/2). As a result, the residual risk of virus transmissionin blood components has declined to approximately 1 in 2,000,000 forHIV, 1 in 2,000,000 for HCV, 1 in 250,000 for hepatitis B virus, and 1in 250,000 to 2,000,000 for HTLV-1 and 2. (Schreiber et al N Engl J Med334:1685-1689; Dodd, Blood safety in the new millennium, Stramer ed,1999, AABB press, Bethesda, Md.)

While the viral safety of the blood supply has dramatically increasedwith the adoption of donor screening and testing measures, the risk ofbacterial sepsis and parasite infection has remained unchanged. Based onthe available evidence, roughly 17% of transfusion related fatalitiesreported to the FDA between 1978 to 1998 were caused by bacterialcontamination. These septic fatalities represent the greatest infectiousdisease risk among transfusion fatalities. (Lee, US FDA BacterialContamination of Platelets workshop, September, 1999) The risk isgreatest from platelet transfusions, with 1 in 60,000 units (Ness etal., Transfusion 41:857-61 (2001)) to 1 in 450,000 units (Kuehnert etal., Transfusion 41:1493-9 (2001)) transfused resulting in fatalities.In addition, there is increasing recognition that parasites other thanmalaria, such as Babesia microti and Trypanosoma cruzi, can betransmitted by blood in the United States. (Dobroszycki et al., JAMA28:927-30 (1999); Herwaldt et al Transfusion 42:1154-8 (2002); Kjemtrupet al Transfusion 42:1482-7 (2002); and Cimo et al., Tex Med 89:48-50(1993)) Although there are less data available, the risk of transmissionof these agents may be as great as or greater than those associated withthe current viral risks for tested agents.

With the reduction of viral risk, the risks of sepsis and parasiteinfection have taken on increased relative importance. Development ofpathogen reduction methods represents an approach to reducetransfusion-transmitted virus, bacteria, and parasite infections. Inaddition, pathogen inactivation may provide an additional layer ofsafety to reduce the residual risk from tested viruses, and maypotentially reduce the transmission of unrecognized or uncharacterizedblood borne agents.

Decontamination treatments that inactivate contaminating pathogens butdo not harm the cellular fractions of blood either are not available orare impractical. Some decontamination treatments include the use ofphotosensitizers, which, in the presence of oxygen and upon exposure tolight that includes wavelengths absorbed by the photosensitizer,inactivate viruses. (EP 0 196 515) Typically, such photochemicals aredyes or other compounds that readily absorb UV or visible light in thepresence of oxygen. These compounds include merocyanine 540 (“MC540”)(U.S. Pat. No. 4,775,625), porphyrin derivatives (U.S. Pat. No.4,878,891), phenothiazine derivatives (U.S. Pat. No. 6,030,767), as wellas other photosensitizers.

Increased virucidal activity of these compounds is realized when theadsorption spectrum of the photosensitizer does not significantlyoverlap the absorption spectra of pigments present in the blood, such ashemoglobin. In order to minimize cellular damage, it is preferable thatthe photosensitizer be nontoxic to the cellular blood components andselectively bind to a component of the virus either that is not presentin red cells or platelets, or, if present therein, that is not essentialto red cells' or platelets' function. It is also preferable if thephotodynamic treatment inactivates extracellular and intracellularviruses as well as proviruses. It is preferable if the photodynamictreatment inactivates bacteria and parasites as well. It is furtherpreferable that the anti-pathogen activity of the photosensitizer is notsignificantly inhibited by the presence of plasma proteins, such ascoagulation proteins, albumin, and the like.

Treatment with known photochemicals, however, frequently does damage tocellular blood components. For example, photochemicals such as theporphyrins (U.S. Pat. No. 4,878,891) and MC 540 (U.S. Pat. No.,4,775,625) cause cellular membrane damage in the presence of light andoxygen that significantly reduces the viability of the phototreated redcells during storage. Similarly, treatment of red blood cells usingphthalocyanine 4 with type 1/type 2 quenchers caused red cell damageeven under optimized conditions, ie., about 2% of the cells hemolyzeafter 21 days of storage. Current FDA guidelines recommend <1% hemolysisafter 6 weeks of storage at 1-6° C. (Transfusion 35:367-70 (1995))Finally, the phenothiazine, dimethylmethylene blue (DMMB), producesroughly 2% hemolysis following 6 weeks of 1-6° C. storage using a redcell storage solution, Erythrosol, that is designed to minimize oreliminate colloidal osmotic hemolysis (Transfusion 42:1200-1205 (2002))With storage solutions that do not protect against colloidal osmotichemolysis, such as ADSOL, roughly 25% of red blood cells hemolyze after6 week refrigerated storage of red cell units treated withdimethylmethylene blue and light (Wagner et al, Transfusion 42:1200-1205(2002))

Colloidal osmotic hemolysis arises from photodamage to the red cellmembrane, which produces ion leakage, and results in an increasedintracellular osmotic pressure at ionic equilibrium due to the osmoticpressure contribution from hemoglobin. This increased intracellularosmotic pressure induces water influx, and ultimately results in cellswelling and hemolysis. (Pooler, Biochim Biophys Acta 812:199-205(1985))

Some residual hemolysis is still observed when red blood cells aretreated with a photodynamic agent even when they are protected fromcolloidal osmotic hemolysis. (Wagner et al., Transfusion 42:1200-1205(2002)) This non-colloidal osmotic hemolysis arises from two sources:from the photodynamic action of red blood cell membrane boundsensitizer, and from photodynamic action of unbound sensitizer. Withdimethylmethylene blue for example, approximately 1.2% hemolysis stillremains after six weeks of 1-6° C. storage even when phototreated redblood cells suspended in Erythrosol to minimize colloidal osmotichemolysis are pre-incubated with a compound, quinacrine, that preventsdimethylmethylene blue binding to the red cell membrane. (Wagner et al.,Photochem Photobiol 76:514-517 (2002))

Known photosensitizers thus induce hemolysis in red blood cells inseveral ways: 1) by producing membrane ion leaks, resulting in colloidalosmotic hemolysis 2) by inducing red blood cell membrane photodamagedifferent from ion leaks which arise from membrane bound sensitizer, and3) by generating red blood cell membrane photodamage different from ionleaks which arise from unbound sensitizer.

Solutions that prolong the shelf life of red cells are known. (e.g.,U.S. Pat. No. 4,585,738) Typically, such solutions contain citrate,phosphate, glucose adenine, and other ingredients and function toprolong shelf life by maintaining the levels of ATP and 2,3-DPD in thecells. In addition, storage or additive solutions that contain highlevels of the impermeable salt, citrate, can protect against colloidalhemolysis from phototreated red cells at ionic equilibrium by creatingan extracellular osmotic pressure equal to the intracellular osmoticpressure arising from hemoglobin. (Transfusion 42:1200-1205 (2002)) Suchstorage solutions are known to those in the art and include ARC-8 andErythrosol. (U.S. Pat. No. 4,585,738 and Vox Sang 65:271 (1993))

Certain antioxidants have been shown to protect cells from photoinducedhemolysis without greatly reducing the level of antiviral activity.However, none has been shown to be useful in the preparation of atransfusible product. For example, the red cell band 3 ligand,dipyridamole, partially protects red cells against dimethylmethyleneblue photoinduced hemolysis by functioning as a red cell specificantioxidant (vanSteveninck et al., Transfusion 40:1330-1336 (2000) andTrannoy et al., Photochem Photobiol 75:167-171 (2002)) None of thosemethods have resulted in the desired level of protection from hemolysis(i.e., <1% hemolysis following 6 weeks of storage).

Despite techniques to minimize colloidal osmotic hemolysis by suspendingred cells in a high citrate containing additive solution, by limitingmembrane damage by preventing sensitizer binding to red cells throughthe use of a competitive binder, or by ameliorating photoinducedhemolysis by adding an antioxidant that specifically binds to red cellmembrane proteins, no method or combination of methods has proved fullysuccessful for decontaminating whole blood, blood components, orcompositions containing concentrated blood components, including highlevels of plasma. There remains, however, an acute need for a safe andeffective method for reducing the level of active pathogeniccontaminants, including viruses and bacteria, in whole blood or bloodcomponents without rendering the blood or blood components unsuitablefor transfusion.

One of the limitations with traditional dyes acting as photosensitizersis that they produce active oxygen species whether or not they are boundto their target. Therefore unbound sensitizer can contribute tophotoinduced collateral damage to the red cell membrane, leading tohemolysis. Therefore, development of a pathogen reduction method tolimit photosensitization from unbound dye may be beneficial to red cellpreservation.

Some dyes have the unusual properties of having a bond linking twoaromatic conjugated double bond systems capable of rotation. Examples ofthese dyes include the methine bond of cyanine dyes and thecarbon-carbon bonds in the 2′, 4′ and 6′ positions of pyrilium dyes.Many cyanine and pyrilium dyes are poor singlet oxygen photosensitizersbecause they can rotate about these carbon-carbon linkages, whichreduces the lifetime of the dye's first excited singlet state, andlimits both the fluorescent quantum yield and the potential forintersystem crossing over to the triplet state necessary for thegeneration of singlet oxygen. However, when these dyes are bound to asubstrate with an orientation that facilitates a prolonged lifetime ofthe first excited state, enhanced fluorescence and enhanced singletoxygen generation can occur. Therefore, pyrilium dyes can be used toeither cause fluorescence or singlet oxygen mediated damage from bounddye without substantial contribution to fluorescence or singlet oxygenproduction from unbound dye. For example, pyrilium dyes have been usedas nucleic acid fluorescent stains where the cell media or buffer doesnot have to be washed because little fluorescence is observed fromunbound dye. (E.g., Nucleic Acids Symp 29:83-84 (1993); and U.S. Pat.No. 6,022,961) Pyrilium dyes have also been described asphotosensitizers for the killing of cancer cells. (U.S. Pat. No.6,242,477) These and all other patents and references cited herein areexpressly incorporated by reference.

Despite the recognition that pyrilium dyes might be employed asphotosensitizers, and observations that unbound pyrilium dyes are poorsinglet oxygen generators, we are unaware of any proposal to use suchcompounds for pathogen reduction in biological fluids such as wholeblood, blood components, or compositions containing concentrated bloodcomponents including high levels of plasma. In addition, one of ordinaryskill in the art would not have been able to predict what class or kindof substituted pyrilium dyes could inactivate pathogens withoutotherwise deleteriously affecting the desired biochemical orphysiological properties of a biological fluid, particularly blood,blood components, and plasma.

SUMMARY OF THE INVENTION

The present invention provides methods for eliminating or diminishingactive pathogenic contaminants, both intracellular and extracellular, inbiological fluids without concomitant loss of desired biochemical orphysiological properties of the fluid. In preferred embodiments, thebiological fluids are selected from whole blood and blood components,including cellular blood components and liquid blood components. Inparticular, the present methods effect decontamination of biologicalfluids by eliminating or diminishing active pathogens such as viruses,bacteria (both gram negative and gram positive) and parasites withoutsubstantial hemolysis.

In one embodiment, the method involves decontaminating a biologicalfluid comprising the steps of: (a) adding a virucidal effective amountof a diphenylpyrilium compound to the biological fluid; and (b)irradiating the resulting mixture with red light for a time sufficientto eliminate or reduce the level of active pathogenic contaminantstherein.

In preferred embodiments, the biological fluids are blood or bloodcomponents including cellular blood components, such as red blood cells(RBC_(S)) and platelets, and liquid blood components, such as plasma, ormixtures of cellular and/or liquid blood components.

As used herein, the term “pyrilium” refers to compounds having thegeneral structure:

wherein Y is O, S, Se, or Te. Compounds useful in the present inventionare pyrilium compounds substituted at the 2, 4, and 6 positions.Preferably, the compounds are diphenylpyrilium compounds.

The term “diphenylpyrilium compounds” or “diphenylpyrilium dyes” refersto 2,4-diphenylpyrilium compounds. Preferably, the phenyl substituentsare further substituted at the para-position with one or more of thefollowing substituents: alkyl, amino, alkylamino, alkoxyamino, aryl,arylamino, arylalkylamino, and arylalkoxyamino. Preferred compounds arethose having the structure:

wherein:

Y is S, Se or Te;

R₁ and R₂ are independently selected from hydrogen, amino, alkylamino,aryl, arylamino, arylalkylamino, and arylalkoxyamino; and

R₃ is hydrogen, alkyl, alkoxy, alkylamino, aryl, arylamino,arylalkylamino, or arylalkoxyamino.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Hemolysis during 1-6° C. storage of RBCs: Control RBCs stored inErythrosol (RAS-2)(open triangles), Control RBCs stored in ADSOL (shadedtriangles); RBCs stored in Erythrosol treated with compound 2 and redlight (open squares); RBCs stored in ADSOL treated with compound 2 andred light (shaded squares); RBCs stored in Erythrosol (RAS-2) andcontaining dipyridamole (DPD) treated with compound 2 and red light(open circles); RBCs stored in ADSOL and containing dipyridamole treatedwith compound 2 and red light (shaded circles). The inset gives anexpanded view of the same data over the 0 to 1% hemolysis range.

FIG. 2. Potassium release during 1-6° C. storage of RBCs: Control RBCsstored in Erythrosol (open triangles), Control RBCs stored in ADSOL(shaded triangles); RBCs stored in Erythrosol treated with compound 2and red light (open squares); RBCs stored in ADSOL treated with compound2 and red light (shaded squares); RBCs stored in Erythrosol andcontaining dipyridamole treated with compound 2 and red light (opencircles); RBCs stored in ADSOL and containing dipyridamole treated withcompound 2 and red light (shaded circles).

FIG. 3 illustrates the structures of thiopyrilium (TP), or2′,4′-bis(4-N,N-dimethylaminophenyl) 6′-methylthiopyrylium iodide, anddiphenylpyrilium (DP).

FIG. 4 illustrates the results from a spectroscopic assay measuring theeffect of DP on TP binding to RBCs suspended in Erythrosol.

FIG. 5 shows the log₁₀ inactivation of virus as a function of TPconcentration.

FIG. 6 shows the effect of dipyridamole and choice of additive solutionon hemolysis following the storage of phototreated RBCs (160 μM TP and1.1 J/cm² light).

FIG. 7 shows the effect of 160 μM TP, 200 μM DP and 1.1 J/cm² light onmorphology score, pH, glucose utilization, lactate production, and ATPlevels of RBCs suspended in Erythrosol in panels A through E,respectively.

FIG. 8 shows the effect of DP on potassium release from RBCs suspendedin Erythrosol and treated with 160 μM TP and 1.1 J/cm² light.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for decontaminating a biologicalfluid comprising: (a) contacting a biological fluid with adecontamination effective amount of a diphenylpyrilium compound; and (b)irradiating the resulting mixture with light of about 560 to about 800nm to achieve a decontaminating effect.

In a preferred embodiment, the invention provides a method fordecontaminating a biological fluid comprising: (a) adding2′,4′-bis(4-N,N-dimethylaminophenyl) 6′-methylthiopyrilium iodide to thebiological fluid to a concentration of about 100 to about 300 μM; (b)adding dipyridamole to the biological fluid to a concentration of about100 to about 300 μM; and (c) irradiating the resulting biological fluidwith light of about 560 to about 800 nm.

The term “decontamination” means both a process whereby the level ofactive pathogen actually present in a given composition is eliminated orreduced, and a process for assuring that a potential pathogeniccontaminant within a composition is below a certain level regardlesswhether such contaminant was ever present in the composition.Decontamination can be effected by rendering pathogens inactive and/ornoninfectious or by reducing the number of pathogens in the composition.A composition containing whole blood or a blood component that has been“decontaminated” can be transfused or manipulated without harming orinfecting anyone exposed thereto.

Preferably, the level of decontamination achieved will be such that theimmune system of the organism exposed to or transfused with thebiological fluid will be capable of overcoming the pathogenic effectthereof, and preventing the onset of any disease associated therewith.It will be understood that, as so defined, the level of decontaminationwill vary depending upon the pathogen.

A decontamination-effective amount of a diphenylpyrilium compound isalso referred to herein as a virucidal effective amount. Adecontamination- or virucidal-effective amount is that capable ofachieving a statistically significant reduction in the level of activepathogenic virus in the biological fluid. Preferably, the virucidaleffective amount is that capable of achieving reduction of at leastabout 4.0 log₁₀ extracellular VSV (Vesicular Stomatitis Virus) in bloodor blood components. More preferably, it is that capable of achieving atleast about 5.0 log₁₀ extracellular VSV reduction and at least about 2.5log₁₀ intracellular VSV reduction. Still more preferably, it is thatcapable of achieving at least about 7.0 log₁₀ extracellular VSVreduction and at least about 5.0 log₁₀ intracellular VSV reduction. Itwill be understood that the amount of diphenylpyrilium compoundnecessary to achieve the desired decontamination will vary from compoundto compound, but that the means for assaying the virucidal effect of thevarious diphenylpyrilium compounds embraced by the claims is well withinthe skill level of one of ordinary skill in the art.

The terms “decontamination effective amount” and “virucidal effectiveamount” also mean an amount sufficient to provide a concentration ofdiphenylpyrilium compound in the biological fluid that is bothacceptable for transfusion and is effective in reducing the level ofactive pathogens in the composition when irradiated with light of anappropriate intensity and wavelength.

As discussed more filly below, the effective concentration ofdiphenylpyrilium compound to be used can be determined empirically byone of ordinary skill in the art. In preferred embodiments, theeffective concentration of diphenylpyrilium compound is about 50 to 300μM, and more preferably 100 to 200 μM.

Preferably, the diphenylpyrilium compound is non-toxic, and theeffective concentration is acceptable for transfusion so that thebiological fluid does not require additional manipulation to remove thediphenylpyrilium compound and thereby risk contamination. Alternatively,the diphenylpyrilium compound concentration in the decontaminatedbiological fluid can be reduced by washing or by adsorption to somebiologically compatible resin.

It will be likewise be understood that such virucidal effective amountswill be effective in achieving statistically significant reductions inactive pathogens other than viruses. As used herein, the term “pathogen”or “pathogenic contaminant” means a contaminant that, upon handling ortransfusion into a recipient is capable of causing disease in thehandler and/or recipient. Examples of pathogenic contaminants include,but are not limited to: viruses, such as retroviruses (e.g. HIV) andhepatitis viruses; bacteria, such as E. coli; parasites, such asTrypanosoma; and leukocytes, such as lymphocytes (which can be areservoir for harboring intracellular viruses).

The term “pathogen” also includes any replicable agent that rnay befound in or infect whole blood or blood components. Such pathogensinclude the various viruses, bacteria, parasites, and leukocytes knownto those skilled in the art to generally be found in or infect wholeblood or blood components. Illustrative examples of such pathogensinclude, but are not limited to: bacteria, such as Streptococcusspecies, Escherichia species, and Bacillus species, viruses, such ashuman immunodeficiency viruses and other retroviruses, herpes viruses,paramyxoviruses, cytomegaloviruses, hepatitis viruses (includinghepatitis B and hepatitis C), pox viruses, and toga viruses; parasites,such as malarial parasites, including Plasmodium species, andtrypanosomal parasites; and leukocytes, such as lymphocytes. The methodsof the present invention afford means for achieving pathogeninactivation corresponding to at least about 6 log₁₀ of bacteria andother pathogens.

As used herein, the term “biological fluid” means fluids of biologicalsignificance or origin including blood or mixtures or suspensionscomprising blood components, milk, tears, saliva, urine, cell culturesupernatants, cell extracts, and cellular supernatant. Preferably, thebiological fluid is blood or blood components. Unless stated otherwise,“blood” refers to mammalian blood.

The term “blood components” means one or more of the constituentcomponents of blood that can be separated from whole blood. The termincludes cellular blood components, such as red blood cells andplatelets; blood proteins, such as blood clotting factors, enzymes,albumin, plasminogen, and immunoglobulins; and liquid blood components,such as plasma and plasma-containing compositions, and mixturescontaining plasma derivatives and/or plasma proteins.

The term “cellular blood component” means one or more components ofwhole blood that comprises cells, such as red blood cells or platelets.

The term “blood protein” means one or more proteins normally found inwhole blood. Illustrative examples of blood proteins found in mammals(including humans) include, but are not limited to, coagulation proteins(both vitamin K-dependent, such as Factor VII or Factor IX, andnon-vitamin K-dependent, such as Factor VIII and von Willebrandsfactor), albumin, lipoproteins (high density lipoproteins and/or lowdensity lipoproteins), complement proteins, globulins (such asimmunoglobulins IgA, IgM, IgG and IgE), and the like.

As used herein, the term “liquid blood component” is intended to meanone or more of the fluid, non-cellular components of whole blood, suchas plasma (the fluid, non-cellular portion of blood of humans or animalsas found prior to coagulation), or serum (the fluid, non-cellularportion of the blood of humans or animals after coagulation).

The term “composition containing the cellular blood component and/or ablood protein” is intended to mean a composition that contains abiologically compatible solution, such as ARC-8 or Erythrosol, and oneor more cellular blood components, one or more blood proteins, or amixture of one or more cellular blood components and/or one or moreblood proteins. Such compositions may also contain a liquid bloodcomponent, such as plasma.

The biological fluids to be decontaminated according to the methods ofthe present invention can be leukodepleted. The term “leukodepleted”means that the concentration of leukocytes in the composition has beenreduced by a specified amount, such as a factor of 10⁵. In preferredembodiments, the biological fluids to be decontaminated in accordancewith the present invention will be first leukodepleted.

The phrase a “transfusible composition” means a composition that can betransfused into the blood stream of a mammal. Transfusible compositionsmight be whole blood or otherwise contain one or more blood components,such as one or more cellular blood components, one or more bloodproteins, and one or more liquid blood components; or mixtures of wholeblood and one or more blood components, such as red blood cells,clotting factors, or plasma.

The ratio of the titer of the control sample to the titer of virus ineach of the treated samples is a measure of viral inactivation. As usedherein, the term “log₁₀ inactivation” is intended to mean the log₁₀ ofthis ratio. Typically, a log₁₀ inactivation of at least about 4indicates that the treated sample has been decontaminated.

The term “fluence” means a measure of the energy per unit area of sampleand is typically measured in joules/cm² (J/cm²). As used herein, theterm “fluence rate” is intended to mean a measure of the amount ofenergy that strikes a given area of a sample in a given period of timeand is typically measured as milliwatts (mW)/cm² or joules/Cm² per unitof exposure.

As used herein, the term “diphenylpyrilium dye” or “diphenylpyriliumcompound” means a compound having the general structure:

When employed in the methods of the present invention, thediphenylpyrilium compound will have one or more amino substituents onone or more of the phenyl groups. Preferred compounds are soluble inpolar solvents, particularly aqueous solvents, and are capable ofpassing through the cell membrane of blood cells in sufficient quantityto reduce the level of active intracellular pathogenic contaminants uponirradiation with light of a suitable intensity and wavelength withoutcausing unacceptable levels of hemolysis. Preferably, the methodachieves the desired decontamination with less than about 5% hemolysis.More preferably, hemolysis resulting from the practice of the presentmethod is less than about 3%, and still more preferably less than about1%.

The unspecified valences of the carbon atoms in the formula above can beoccupied by hydrogen or by any organic or inorganic moiety that does notadversely affect the amphiphilic character of the diphenylpyriliumcompound.

One skilled in the art can determine the suitability of a particularsubstituent group or groups empirically using standardized assays fordetermining the level of active intracellular and extracellularpathogenic contaminants and standardized assays for determininghemolysis levels. As used herein, hemolysis is measured after 42 daysstorage at 1-6° C.

Illustrative examples of suitable substituents include, but are notlimited to, alkyl groups, alkenyl groups, allynyl groups, hydroxylgroups, alkoxy groups, aryl groups, heteroaryl groups, aryloxy groups,heteroaryloxy groups, nitro groups, amine groups, amide groups,alkylcarboxyl groups, arylhaloalkyl groups haloaryl groups. Preferredorganic moieties include alkyl groups, such as methyl, ethyl and propyl;alkenyl groups such as ethenyl; alkynyl groups such as acetenyl; andamines such as methylamine and dimethylamine.

The term “leukocyte depleted blood component” is intended to mean ablood component, such as plasma, as defined above that has been filteredthrough a filter that depletes the concentration of leukocytes in theplasma by a factor as least 10³. Such filters are identified by the logof the factor by which the blood component is depleted of leukocytes.

The term “extracellular pH” means the pH of the liquid medium in whichcellular blood components, such as red blood cells, are stored ormaintained.

The term “a biologically compatible solution” is intended to mean anaqueous solution to which cellular blood components can be exposed, suchas by being suspended therein, and remain viable, i.e., retain theiressential biological and physiological characteristics.

Preferably, such biologically compatible solutions contain an effectiveamount of at least one anticoagulant. Preferred biologically compatiblesolutions in the context of this invention protect against colloidalosmotic photoinduced hemolysis. One method for achieving this is by theaddition of citrate at concentrations that balance the osmotic pressurecontributed by hemoglobin.

The term “a biologically compatible buffered solution” is intended tomean a biologically compatible solution having a pH and osmoticproperties (e.g., tonicity, osmolality and/or oncotic pressure) suitablefor maintaining the integrity of the cell membrane of cellular bloodcomponents. Suitable biologically compatible buffered solutionstypically have a pH between 5 and 8.5 and are isotonic or onlymoderately hypotonic or hypertonic. Biologically compatible bufferedsolutions are known and readily available to those of skill in the art.Illustrative examples of suitable solutions include, but are not limitedto, those listed in Table 1 below showing the substances present inanticoagulant solution into which whole blood is drawn, and thesubstances present in the additive solution added after whole blood iscentrifuged and plasma removed to make packed red cells. Additivesolutions containing citrate such as Nutricell and Erythrosol arepreferred because these solutions protect against 10 colloidal osmotichemolysis, whereas those lacking citrate such as ADSOL do not. TABLE 1Anticoag. (70 mL) CPD¹ CP2D CPD Dextrose (hydrous) 1.785 g  3.57 g 1.785g Sodium citrate  1.84 g  1.84 g  1.84 g Citric acid 0.229 g 0.229 g0.229 g Sodium phosphate 0.155 g 0.155 g 0.155 g monobasic (dihydrate)Additive soln (110 mL) ADSOL Nutricell Erythrosol² Dextrose (hydrous) 2.42 g  1.21 g  0.99 g Sodium chloride 0.990 g 0.451 g 0.860 g Adenine0.0297 g  0.033 g 0.0237 g  Mannitol 0.825 g — 0.851 g Sodium citrate(dihydrate) — 0.647 g  0.86 g Citric acid — 0.0462 g  — Sodium phosphate— 0.304 g 0.0711 g  monobasic (dihydrate) Sodium phosphate dibasic — —0.257 g (dihydrate)¹CPD = citrate, phosphate, dextrose; CP2D = CPD having twice theconcentration of dextrose.²Also referred to as RAS-2 (Red Cell Additive Solution No.2).

In certain preferred embodiments, whole blood is first drawn from adonor into a suitable biologically compatible buffered solutioncontaining an effective amount of at least one anticoagulant. Suitableanticoagulants are known to those skilled in the art, and include, butare not limited to, lithium, potassium or sodium oxalate (15 to 25 mg/10mL of blood), sodium citrate (40 to 60 mg/10 mL blood), heparin sodium(2 mg/10 ml of blood), disodium EDTA (10 to 30 mg/10 mL of blood) orACD-Formula B solution (1.0 mL/10 mL blood).

The whole blood so collected can be decontaminated according to themethods of the present invention. Alternatively, the whole blood can beseparated into blood components, including, but not limited to plasma,platelets and red blood cells, by any method known to those of skill inthe art. For example, blood can be centrifuged for a sufficient time andat a sufficient centrifugal force to sediment the red blood cells.Leukocytes collect primarily at the interface of the red cells and theplasma-containing supernatant in the buffy coat region. The supernatant,which contains plasma, platelets, and other blood components, can beremoved and centrifuged at a higher centrifugal force, whereby theplatelets sediment.

Human blood normally contains about 3×10⁹ leukocytes per 500 mL of wholeblood (1 unit). The concentration of leukocytes, which sediment with thered cells, can be decreased if desired by passing through a filter thatdecreases leukocyte concentration by selected orders of magnitude.Leukocytes can also be removed from each of the components by filtrationthrough an appropriate filter that removes them from the solution.

In one preferred embodiment, the whole blood or blood component to bedecontaminated is obtained in, prepared in, or introduced into, gaspermeable blood preservation bags, which are sealed and flattened to awidth sufficiently narrow to permit light to irradiate the contents,such that any pathogenic contaminant present in the blood or bloodcomponent in the bag will be irradiated. Conventional blood bags used inthe art can be used provided the bag is transparent to the selectedwavelength of light.

In an alternative preferred embodiment, blood can be passed from one bagthrough tubing into another bag, which serves as a flow cell, and isflattened to a width sufficiently narrow to permit light to irradiatethe flow cell contents, such that any pathogenic contaminant present inthe blood or blood component in the bag will be irradiated, and theirradiated blood is subsequently passed through tubing into a receivingblood bag.

Optionally, the gas permeable blood preservation bag also containsoxygen. While not wishing to be bound by any theory of operability, itis believed that certain species of amphiphilic diphenylpyriliumcompound employed in the methods of the invention, in addition tointercalating between base pairs of DNA, generate singlet oxygen whenirradiated with light of an appropriate wavelength. As is known to thoseskilled in the art, singlet oxygen directly or products thereof (e.g.,superoxides, hydroxy radicals, etc.) cause pathogen inactivation.Accordingly, it is preferred that, at least for certain species ofamphiphilic diphenylpyrilium compounds, the composition beingdecontaminated contain a suitable amount of oxygen.

The composition that is to be decontaminated may also include anysuitable biologically compatible buffer known to those of skill in theart. Examples of such buffers include, but are not limited to,AC2D/Nutricell and ACD/Erythrosol. In a preferred embodiment of thisinvention, the biologically compatible buffer is ACD/Erythrosol.

The irradiation step can be performed in any fashion that ensures thatthe diphenylpyrilium compound is thoroughly distributed throughout thebiological fluid and is exposed to sufficient light to achieve thedesired decontamination effect. Preferably, the irradiation step isperformed on a thin layer or film of the biological fluid.Alternatively, the irradiation step can be performed on the biologicalfluid in a conventional vessel with appropriate stirring or agitation toeffect thorough irradiation throughout the mixture. One of ordinaryskill in the art will appreciate that such alternative embodiments mightrequire light of greater energy to effect the desired level ofirradiation.

Although it will be understood that the parameters can be varied,exemplary irradiation conditions are those wherein the thin film is of athickness of about 0.5 mm to about 3 mm, and more preferably about 1 mm.The film is irradiated with light of wavelength of about 560 to about800 nm, preferably about 590 to about 640 nm; and still more preferablyabout 620 nm. One of ordinary skill in the art will appreciate that asone deviates from light of the optimal wavelength, greater amounts ofenergy may be required to achieve the same decontamination effect.

Irradiation of sufficient energy is effected to achieve the desiredlevel of decontamination. Generally, irradiation of at least about 0.025j/cm² of light of about 560 to about 800 nm is effected; and preferably,irradiation of about 0.05 to about 5.0 J/cm² of light of about 560 toabout 800 nm is effected. More preferably, irradiation of about 0.1 toabout 1.0 j/cm² of light of about 590 to about 640 nm is effected. Stillmore preferably irradiation of about 0.1 to about 0.4 J/cm² of 620 nmlight (which corresponds to about 1.1 to about 2.2 J/cm² of 670 nmlight) is effected.

The preferred amphiphilic diphenylpyrilium compounds employed in themethods of the present invention include those of the formula:

wherein: each of R₁, R₂, and R₃ is independently selected from the groupconsisting of an alkyl group, an alkenyl group, an alkynyl group, analkoxy group, hydroxyl, amino, alkylamino, aryl, arylamino,arylalkylamino, arylalkoxyamino (e.g., phenylmorpholino) and hydrogen;and Y is sulfur or selenium. In preferred embodiments, Y is sulfur orselenium; R₁ and R₂ are independently selected from hydrogen, amino,alkylamino(monalkylamino and dialkylamino) and alkoxyamino (includingheterocycles incorporating oxygen and/or nitrogen within the ring, e.g.,morpholino); and R₃ is hydrogen, alkyl, alkoxy, aryl, arylamino,arylalkylamino, or arylalkoxyamino.

The term “alkyl group” means a straight or branched chain hydrocarbonradical having from 1-10 carbon atoms; preferably 1 to 6 carbon atoms;and more preferably 1 or 2 carbon atoms.

The term “alkenyl group” means a straight or branched chain hydrocarbonradical having 2-10 carbon atoms and at least one carbon-carbon doublebond.

The term “alkynyl group” means a straight or branched chain hydrocarbonradical having 2-10 carbon atoms and at least one carbon-carbon triplebond.

The term “axyl group” means a cyclic aromatic hydrocarbon radical havingfrom 6-12 carbon atoms; preferably 6-10 carbon atoms; and includesgroups such as phenyl, naphthyl, and the like.

The term “aralkyl group” means a straight or branched chain hydrocarbonradical having from 1 to 6 carbon atoms bound to a cyclic aromatichydrocarbon radical having from 6-12 carbon atoms in the ring(s), andincludes radicals such as benzyl, 2-phenylethyl and the like.

The term “heteroaryl group” is intended to mean a monocyclic or bicyclicaromatic radical having from 4-11 carbon atoms and at least oneheteroatom (i.e. an oxygen atom, a nitrogen atom and/or a sulfur atom)in the ring(s), such as thienyl, fulryl, pyranyl, pyridyl, quinolyl andthe like.

In a preferred embodiment, R₁ and R₂ are independently selected from thegroup consisting of: amino, monomethylamino or dimethylamino. In a morepreferred embodiment, R₁ and R₂ are both dimethylamino.

In a preferred embodiment, R₃ is alkyl, alkoxy, or aryl. In a morepreferred embodiment, R₃ is alkyl of 1-6 carbons or phenyl. In a stillmore preferred embodiment, R₃ is methyl. The term alkoxy refers to analkyl ether wherein the alkyl group is as defined above.

The amphiphilic diphenylpyrilium compounds used in the methods of thepresent invention can be prepared according to methods and techniquesknown to those of ordinary skilled in the art. Suitable syntheticmethods for the preferred compound are described, for example, in U.S.Pat. No. 6,022,961, which is incorporated herein by reference.

In a particularly preferred embodiment of the present invention,2′,4′-bis(4-N,N-dimethylaminophenyl) 6′-methylthiopyrilium iodide

is employed as the amphiphilic diphenylpyrilium dye. Preferably, the2′,4′-bis(4-N,N-dimethylaminophenyl) 6′-methylthiopyrilium iodide isintroduced into the whole blood or blood component to be decontaminatedat a concentration of about 100 to 200 μM.

The mixture of the whole blood and/or blood components and amphiphilicdiphenylpyrilium compound is then irradiated with light of anappropriate wavelength (or a mixture of wavelengths) and intensity. Asused herein, the term “appropriate wavelength and intensity” is intendedto mean light of a wavelength and intensity that can be absorbed by thediphenylpyrilium compound, but does not damage the blood or bloodcomponents present. It is within the level of ordinary skill in the artto select such wavelength and intensity empirically based on certainrelevant parameters, such as the particular compound employed and itsconcentration in the composition. For example, one having skill in theart would appreciate that if the intensity of the light source isdecreased, a greater concentration of diphenylpyrilium compound and/orlonger exposure time could offset the decrease in intensity. Likewise,the use of light of less optimal wavelength can be offset by increasingthe radiant energy.

An appropriate wavelength is preferably selected based on the absorptionprofile of the diphenylpyrilium compound employed, and is mostpreferably one that does not result in substantial damage to one or moreof the cellular blood components in the composition beingdecontaminated.

Known model viral systems can be used to test the selected dye and thelight source for efficacy. Model viral systems include, but are notlimited to, vesicular stomatitis virus (“VSV”: an animal virus thegenome of which is encoded in single stranded RNA), and Pseudorabiesvirus (an animal virus that contains its genome in double stranded DNA).Based on the effective values of parameters such as wavelength and lightintensity measured for such model systems, one of skill in the art canroutinely select suitable values for these parameters for use inpractice of the present invention.

In a preferred embodiment of this invention, oxygenated red blood cells,which have been leukodepleted with a five log filter, are firstsuspended in Erythrosol or Nutricell at a hematocrit of about 15 toabout 50 percent, dipyridamole is added at a final concentration ofabout 50 to 300 μM, and 2′,4′-bis(4N,N-dimethylaminophenyl)6-methylthiopyrilium iodide is added to a final concentration of about100 to 200 μM. The blood is placed in a flattened container to produce athin film. Preferably, the thin film is of a thickness of about 0.5 mmto about 3 mm, and more preferably about 1 mm. This film is irradiatedwith red light of wavelength of about 560 to about 800 nm at sufficientenergy to reduce the level of active pathogenic contaminant in theblood.

In other embodiments of this invention, biological fluids containingplatelets and fluids containing high concentrations of plasma can bedecontaminated by contact with an effective amount of an amphiphilicdiphenylpyrilium compound for sufficient time plus irradiation withlight of an appropriate wavelength and intensity.

Following decontamination in accordance with the methods of thisinvention, the biological fluid can be stored or transfused inaccordance with conventional practice. Alternatively, for fluids such asred cell preparations or platelet rich plasma, the decontaminated fluidcan be centrifuged at a force sufficient to produce a pellet of thecellular components. The supernatant can be removed followingcentrifugation and the cells resuspended to reduce the concentration ofresidual photosensitizer and any reaction products.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention.

All patents and publications referred to herein are expresslyincorporated by reference.

EXAMPLE 1

Compounds 1-26 in Table 2 were screened for virucidal and photohemolyticactivity. Plasma containing red cells were oxygenated by gas overlay,leukodepleted by a 5 log₁₀ filter, suspended in Erythrosol to ahematocrit of 20%, and deliberately inoculated with extracellular VSV.Various concentrations of a compound were added to the oxygenated,leukodepleted cell suspension, and a 1 mm film of the suspension wassubsequently illuminated for 2 minutes with 8.9 mW/cm² of red light (670nm [peak intensity]±13 nm [half peak intensity]). Results in Table 2correspond to compounds of Formula I. TABLE 2* Formula I

Compound # Y R₁ R₂ R₃ 1 O N(CH₃)₂ N(CH₃)₂ CH₃ 2 S N(CH₃)₂ N(CH₃)₂ CH₃ 3Se N(CH₃)₂ N(CH₃)₂ CH₃ 4 Te N(CH₃)₂ N(CH₃)₂ CH₃ 5 S N(CH₃)₂ N(CH₃)₂ φ 6Se N(CH₃)₂ N(CH₃)₂ φ 7 S N(CH₃)₂ N(CH₃)₂ φ-N(CH₃)₂ 8 Se N(CH₃)₂ N(CH₃)₂φ-N(CH₃)₂ 9 Se NH₂ N(CH₃)₂ φ-NH₂ 10 S NH₂ N(CH₃)₂ φ 11 Se NH₂ N(CH₃)₂ φ12 S NH₂ morpholino φ- morpholino 13 Se NH₂ morpholino φ- morpholino 14S NH₂ morpholino φ-NH₂ 15 Se NH₂ morpholino φ-NH₂ 16 S NH₂ H φ-morpholino 17 Se NH₂ H φ- morpholino 18 S morpholino morpholino φ-morpholino 19 Se morpholino morpholino φ- morpholino 20 S NH₂ N(CH₃)₂ φ-morpholino 21 Se NH₂ N(CH₃)₂ φ- morpholino 22 S NH₂ morpholino φ 23 SeNH₂ morpholino φ 24 Te NH₂ N(CH₃)₂ φ 25 Te N(CH₃)₂ N(CH₃)₂ φ-N(CH₃)₂ 26Se N(CH₃)₂ H φ*In Table 2, the symbol “φ” denotes a phenyl group.

TABLE 3 Log₁₀ VSV log₁₀ VSV inact. inact. Compound Concentration (extra-(intra- Hemolysis (%) # (μM) cellular) cellular) (day 42) 1 20 <0.5 40<0.5 80 1.3 160 2.1 2 10 1.3 20 2.2 30 2.7 60 4.8 90 5.7 160 >7 >5 0.453 10 0.9 30 2.5 60 4.2 90 4.6 120 5.6 4 20 <0.5 60 <0.5 120 <0.5 180<0.5 240 0.5 5 1 3.1 5.2 5 5.9 2.3 5.1 10 7.9 2.6 7.3 15 >7.9 3.7 6.425 >8.4 4.7 6.5 6 1 5 10 >7.9 2.5 17.1 15 >8.1 10.5 25 >8.0 10.8 7 1 0.55 1.0 10 2.1 15 3.0 25 3.4 3.2 (day 7) 8 1 3.6 2.4 (day 7) 5 7.4 30.210 >8.3 4.7 42.2 15 >8.3 25 >8.3 9 1 0.6 5 1.6 10 2.3 15 2.7 25 3.3 503.9 0.83 50 μM + 6.9 28.3 2.2 J/cm² 10 1 0.7 5 2.2 10 3.8 15 4.5 25 5.60.4 40 5.1 0.3 40 μM + 7.8 1.1 0.46 2.2 J/cm² 11 1 1.9 5 4.6 10 6.2 1.715 7.4 2.1 25 >8.2 2.4 2.5 12 25 2.2 1 0.9 5 1.5 10 1.7 15 2.0 25 2.20.19 13 1 0.9 5 2.6 10 3.3 15 4 25 4.5 35 4.7 0.6 35 μM + 7.0 5.4 2.2J/cm² 14 1 <0.5 5 <0.5 10 <0.5 15 <0.5 25 <0.5 15 1 <0.5 5 <0.5 10 <0.515 <0.5 25 <0.5 16 1 <0.5 5 <0.5 10 <0.5 15 <0.5 25 <0.5 17 1 0.7 5 1.210 1.7 15 2.1 25 1.7 18 1 <0.5 5 <0.5 10 0.7 15 1.0 25 1.2 4.3 (day 7)19 1 <0.5 5 <0.5 10 <0.5 15 <0.5 25 1.3 0.78 (day 7) 20 1 0.8 5 1.8 102.5 15 3.4 25 4.0 40 4.8 0.9 40 μM + 7.1 7.1 2.2 J/cm² 21 1 1.6 5 4.1 105.3 3.4 15 5.7 3.8 25 7.8 3.8 22 1 <0.5 5 <0.5 10 <0.5 15 <0.5 25 1.4 231 0.7 5 1.8 10 3.4 15 4.0 25 4.8 30 5.7 0.5 30 + 8.2 3.5 2.2 J/cm² 24 1<0.5 5 <0.5 10 <0.5 15 <0.5 25 1.5 4.9 25 1 <0.5 0.31 5 <0.5 0.50 10<0.5 0.56 15 1.1 0.77 25 2.1 2.97 26 1 <0.5 5 <0.5 10 <0.5 15 <0.5 25<0.5

As shown in Table 3, compound 2, a diphenylpyrilium dye, inactivates>7log₁₀ of extracellular VSV and >5 log₁₀ of intracellular VSV withoutcausing undue hemolysis during 42 day 1-6° C. storage of red cellssuspended in Erythrosol.

EXAMPLE 2

Compound 2 was screened for bacteridcidal activity. Plasma containingred cells were oxygenated by gas overlay, leukodepleted by a 5 log₁₀filter, suspended in Erythrosol to hematocrit of 20%, and inoculatedwith high levels of an organism to yield final bacterial counts rangingfrom 106 to 108 CFU/mL. Compound 2 was added to the deliberatelycontaminated, oxygenated, leukodepleted cell suspension to give a finalconcentration of 160 μM, and a 1 mm film of the suspension wassubsequently illuminated for 2 minutes with 8.9 mW/cm² of red light (670nm [peak intensity]±13 nm [half peak intensity]). Results are shown inTable 4. TABLE 4 Organism log₁₀ inactivation E. coli >7.9 S.marcescens >6.9 Y. enterocolitica >7.5 D. radiodurans >7.9 S.epidermidis >7.9 S. aureus >6.8 P. fluorescens >7.7 S. liquefaciens >7.4

Both gram positive and gram negative organisms were inactivated bycompound 2 and light to the limit of detection (<1 CFU/mL) usinginoculula >6.8 log₁₀.

EXAMPLE 3

The effect of different additive solutions and dipyridamole onphotoinduced hemolysis by compound 2 was studied. Plasma containing redcells were oxygenated by gas overlay, leukodepleted by a 5 log₁₀ filter,suspended in either Eiytlrosol or ADSOL additive solution to ahematocrit of 20%. Dipyridamole was added to some of the Erythrosol orADSOL red cell suspensions to a final concentration of 200 μM. Compound2 was then added to some of the Erythrosol or ADSOL red cellsuspensions, some of which contained dipyridamole, to a finalconcentration of 160 μM. For suspensions containing compound 2, a 1 mmfilm of the suspension was subsequently illuminated for 2 minutes with8.9 mW/cm² of red light (670 nm [peak intensity]±13 nm [half peakintensity]). Red cell suspensions were concentrated to 45% hematocrit bycentrifugation. Untreated and phototreated red cell suspensions werestored for up to 42 days at 1-6° C. and assayed for hemolysis. Resultsare shown in FIG. 1. The data demonstrate, 1) that <1% hemolysis isobserved in Erytlrosol containing RBC suspensions treated with compound2 and light and 2) that less hemolysis is observed in RBC suspensionsthat contain dipyridamole than those that lack dipyridamole.

EXAMPLE 4

The effect of different additive solutions and dipyridamole on compound2 photoinduced red cell potassium leakage was studied. Plasma containingred cells were oxygenated by gas overlay, leukodepleted by a 5 log₁₀filter, and suspended in either Erythrosol or ADSOL additive solution toa hematocrit of 20%. Dipyridamole was added to some of the Erythrosol orADSOL red cell suspensions to a final concentration of 200 μM. Compound2 was then added to some of the Erythrosol or ADSOL red cellsuspensions, some of which contained dipyridamole, to a finalconcentration of 160 μM. For suspensions containing compound 2, a 1 mmfilm of the suspension was subsequently illuminated for 2 minutes with8.9 mW/cm² of red light (670 nm [peak intensity]±13 nm [half peakintensity]). Red cell suspensions were concentrated to 45% hematocrit bycentrifugation. Untreated and phototreated red cell suspensions werestored for up to 42 days at 1-6° C. and assayed for extracellularpotassium. Results are shown in FIG. 2. Data show that the addition ofdipyridamole reduces potassium leakage of Erythrosol or ADSOL red cellsuspensions during storage.

EXAMPLE 5

Plasma containing red cells were oxygenated by gas overlay,leukodepleted by a 5 log₁₀ filter, and suspended in Erythrosol to ahematocrit of 20%, and deliberately inoculated with either intracellularor extracellular VSV. Dipyridamole was added to the oxygenated,leukodepleted red cell suspension at a final concentration of 200 μM.Compound 2 was then added to the red cell suspension at a finalconcentration of 160 μM, and a 1 mm film of the suspension wassubsequently illuminated for 2 minutes with 8.9 mW/cm² of red light (670nm [peak intensity]±13 nm [half peak intensity]). Samples weresubsequently assayed for plaque forming ability. In the presence ofdipyridamole, >7 log₁₀ inactivation of extracellular VSV and 4.0 log₁₀of intracellular VSV was demonstrated.

EXAMPLE 6

Red Blood Cell (RBC) preparation and oxygenation Packed RBCs wereprepared from units of whole blood (500-50 mL) collected in 70 mL CDP intriple-pack container systems (PL146 primary container, BaxterHealthcare, Deerfield, Ill.) by the American Red Cross, Research BloodDepartment, Holland Laboratory for the Biomedical Sciences. Units werecooled to to 6° C. overnight, centrifuged at 1471×g for 4 minutes, andplatelet-rich plasma and buffy coat were removed. The packed RBCs werediluted to an hematocrit (hct) of approximately 50% with cold Erythrosol(Hogman C F, Eriksson L, Gong J, Hogman A B, Vikholm K, Debrauwere J,Payrat J M, Stewart M. Half-strength citrate CPD combined with a newadditive solution for improved storage of red blood cells suitable forclinical use, Vox Sang. 1993;65(4):271-8) or, when noted, with coldADSOL (Baxter Healthcare); subsequently white cell reduced by using afilter (Leukotrap-SC RC, Pall Medical, East Hills, N.Y.); and oxygenatedby adding 230 mL of a 60 to 40 percent O₂ to N₂ gas mixture to 150 mL ofa RBC suspension in a 600 mL container (PL146 plastic, BaxterHealthcare) and by subsequent incubation for 30 minutes at 10 to 6° C.with agitation (orbital shaker, 100 r.p.m., 19-mm orbit, VWR Scientific,West Chester, Pa.). Oxygen levels were measured by use of a blood gasanalyzer (Rapidalab 348, Bayer Corp., Medfield Mass.) and were routinelysupersaturated with levels greater than 400 mm Hg.

TP Preparation

The structures of TP, or 2′,4′-bis(4-N,N-dimethylaminophenyl)6′-methylthiopyrylium iodide, and DP are given in FIG. 3. TP wassynthesized accord to the method described by Yamamoto and colleagues(Yamamoto N, Okamoto T, Miyazaki T, Kawaguchi M. Fluorescent staincontaining pyrylium salt and fluorescent staining method of biologicalsample. U.S. Pat. No. 6,022,961). The dye was purified by mediumpressure (100-psi) liquid chromatography. A gradient of methylenechloride:methanol (100:0-94:6) was used as an eluent for dye bound tosilica gel (40μ, Scientific Absorbents, Atlanta, Ga.). The compound washomogeneous by thin layer chromatography. NMR analysis revealed thecompound to be 90% pure, with 10% of the compound being the pyryliumprecursor dye where an oxygen atom substitutes for sulfur in the centralring. Pathogen reduction experiments with purified pyrylium precursorrevealed that the impurity possessed approximately one-third thephotoactivity of TP at the same dye concentration (data not shown).

Addition of Virus or Bacteria and TP Phototreatment

We added stock cultures of extracellular and intracellular viruses orbacteria to oxygenated, white cell reduced, RBCs at approximately 50percent hct. A 10 mM DP stock solution, a 500 μM freshly prepared stocksolution of TP, each in Erythosol or ADSOL as noted in figures, and theappropriate additional additive solution were added sequentially toyield 200 μM DP and the desired TP concentration in the final 20% hctRBC suspension, which was utilized to improve light transmission,thereby maximizing pathogen reduction. The volume of the pathogen spikerepresented <10 percent of the total volume of the 20% hct RBCsuspension. The suspension was thoroughly mixed and divided into 2 mLaliquots in polystyrene culture dishes (50 mm bottom diameter) toproduce a 1 mm blood film. All treated and control samples contained DPand TP but control samples were not illuminated. We agitated culturedishes at room temperature on a horizontal reciprocal shaker (70cycles/min) for 15 minutes in the dark prior to illumination.

Because RBC storage studies required greater volumes that virus orbacterial studies, 45 petri dishes containing 2 mL each of 20% hct RBCswith DP and TP were illuminated with agitation as described above, theircontents pooled, concentrated to 45% hct by centrifugation andresuspension in the appropriate additive solution, and transferred to a150-mL PL145 container to provide sufficient volume for RBC storagestudies. RBC suspensions prepared at 45-percent hct that did not containDP or TP and were not illuminated served as controls. In some red cellstorage experiments, identically prepared RBC samples that contained TPbut did not contain DP were illuminated to determine the protectiveeffect of DP and are noted in the text and figures.

Illumination was carried out using a red LED source (Q-beam 2001-MED,Quantum Devices, Inc., Barneveld, Wis.), which emitted 670 (peakintensity)±13 nm (half peak intensity) light with fluence ratesadjustable up to 9.0 mW/cm². Fluence rates were measured by use of ahandheld laser power meter with a silicon cell sensor (EdmundsIndustrial Optics, Barrington, N.J.). All phototreated samples wereexposed 2 minutes to the 9.0 mW/cm² source, corresponding to a 1.1 J/cm²light exposure.

Virus Assays

Source of virus, bacteria and infected host cells. VSV was provided byMed Lieu (Hyland Diagnostics, Duarte, Calif.). BVDV was purchased fromthe American Type Culture Collection, Manassas, Va.). PRV was providedby Shirley Mieka (American Red Cross, Rockville, Md.). DHBV, HIV-1 IIIB,and an HIV-1 infected HUT 78 permissive B-cell line, BP-1 (originallyisolated by Bernard Poiesz using the method of Federico M, Titti F,Butto S, Orecchia A, Carlini F, Taddeo B, et al., Biologic and molecularcharacterization of producer and nonproducer clones from HUT-78 cellsinfected with a patient IV isolate, AIDS Res Hum Retroviruses,1989;5:385-96), was obtained from M. Khalid Ijaz (MicroBioTest,Sterling, Va.). Clinical strains of E. coli, P. fluorescens wereprovided by Joseph Campos (Childrens' National Medical Center,Washington, D.C.). Clinical strain of S. marcescens and Y.enterocolitica (serotype O:3) was provided by Vince Piscitelli (Yale NewHaven Hospital, New Haven, Conn.). S. epidermidis (ATCC #1228), S.aureus (ATCC #27217), and S. liquifaciens (ATCC #27529) were purchasedfrom the American Type Culture Collection, Manassas, Va.

Mammalian virus assays. We propagated VERO (isolated from African greenmonkey kidney, CCL81, ATCC) and MDBK (CRL6071, ATCC) cells in medium(RPMI 1640 supplemented with glutamine, Biofluids, Rockville, Md.)supplemented with 10-percent bovine serum. Cells were seeded intosix-well culture plates and allowed to grow to confluency. Control andphototreated samples were serially diluted 10-fold, plated ontoconfluent VERO (for VSV and PRV) or MDBK (for BVDV) cell monolayers, andincubated for 1 hour with gentle rocking at 37° C. for virus adsorptionto cells. The inoculum was removed by aspiration and washed with PBS, asemi-liquid agar layer (0.2-percent) was added to each well and infectedmonolayers were incubated at 37° C. in air containing 5-percent CO₂.Incubation periods were: VSV, 1 day; PRV, 2 to 3 days; BVDV, 5 to 6days. After incubation, the agar layer was removed by aspiration and themonolayer was stained with 0.1-percent crystal violet in ethanol for atleast 15 minutes. The stain was removed by aspiration, the plates werewashed with water, and the plaques enumerated.

The isolation and culture of primary duck hepatocytes from <1-week-oldseronegative White Pekin ducklings (Anas domesticus) and DHBVimmunofluorescence assay was performed by MicroBioTest (Sterling, Va.)according to a previously published procedure (Wagner S J, SkripchenkoA, Pugh J C, Suchmann D B, Ijaz M, Duck hepatitis B photoinactivation bydimethylmethylene blue in RBC suspensions, Transfusion, 2001;41:1154-8).Treated and control DHBV-spiked RBC samples were serial diluted 1 in 10in L-15 medium (BioFluids) and 1-mL volumes were subsequently inoculatedin primary duck hepatocytes monolayers in quadruplicate. The infectedcultures were incubated at 37° C. with 5-percent CO₂ overnight for virusattachment and entry. The inoculum was then removed, cell monolayerswere washed once with complete L-15 medium to remove excess RBCs, andthen each well was overlaid with approximately 2 mL of fresh L-15medium. Infected monolayers were incubated an additional 6 to 7 days at37° C., with media changes every 2 days. After incubation, the mediumwas removed by aspiration, monolayers were washed with PBS and removedby aspiration, and monolayers were subsequently fixed by incubation with1 to 2 mL of −20° C. ethanol for 2 hours at 4° C. The ethanol wasremoved by aspiration, washed with PBS, and incubated at roomtemperature for at least two hours with 0.25 mL of a 1 in 2-mL dilutionof DHBV MoAb directed against the pre-S domain of the DHBV envelope(Pugh J C, Di Q, Mason W S, Simmons H, Susceptibility to duck hepatitisB virus infection is associated with the presence of cell surfacereceptor sites that efficiently bind virus particles, J Virol1995;69:4814-22). The antibody was removed by aspiration, washed withPBS, aspirated, and incubated for 2 hours at room temperature with 0.25mL of a 1-in-200 dilution of goat anti-mouse IgG-FITC conjugate (JacksonImmuno-Research Laboratories, West Grove, Pa.). The secondary antibodieswere removed by aspiration, and the fluorescence-stained monolayer waswashed with PBS and aspirated. Monolayers were examined by UV lightfluorescent microscopy (Diaphot, Nikon, Columbia, Md.) and were scoredpositive if wells contained one or more DHBV surface-antigen-positivehepatocytes. Virus titers were determined by the median tissue cultureinfective dose method (Reed L J, Muench H A, A simple method ofestimating fifty percent end points, Am J Hyg 1938;27:493-7).

Titration of extracellular and intracellular HIV-1 was carried out byMicroBioTest, Sterling, Va. Control and phototreated RBCs containingextracellular or intracellular HIV-1 were serially diluted 10-fold inRPMI1640 supplemented with glutamine (ATCC, Manassas, Va.) andcontaining 10% fetal bovine serum (Invitrogen, Calif.), and 0.5 mL ofeach dilution of control or phototreated sample was transferred into24-well plates (Corning, Acton, Mass.) in quadruplicate. To each ofthese wells was added 1 mL of a T-cell lymphoblastic host cell line,CCRF-CEM, for coculture (Yamada O, Hattori N, Kurimura T, Kita M,Kishida T, Inhibition of growth of HV by human natural interferon invitro, AIDS Res Hum Retroviruses, 1988;4:287-94). Virus and host cellswere incubated at 37° C. in 5% CO₂ in air for 18-24 hours for virusadsorption, and then one-half of the cell suspension was replaced withfresh medium. Infected cells were incubated an additional 3-4 weeks witha replacement of one-half of the volume of the supernatant with freshmedium 3 times per week. After 9-12 additions of fresh media, we assayedculture fluid by HIV-1 p24 antigen enzyme-linked immunosorbent assay(Zepto Matrix, Buffalo, N.Y.).

Bacterial Assays

We prepared fresh overnight cultures of bacteria by inoculatingsingle-colony isolated into Luria broth (Becton Dickinson, Cockeysville,Md.). Cultures were incubated under aerobic conditions at 30 or 37° C.,depending on the strain. Following inoculation into RBC suspensions,bacterial counts were determined in phototreated and control samples by1-in-100 serial dilution of fully mixed samples in unbuffered saline,adding either 0.1 or 1.0 mL of the diluted or neat suspension,respectively, to 3 mL of 0.8-percent molten agar (43° C.) and pouringthe molten agar over Luria broth agar plates. We counted colonies afterincubation for 24-72 hours at 30 or 37° C., with time and incubationtemperature depending on the strain. Colonies were counted from allplates that contained between 1 and 750 colonies per plate.

RBEC Assays

We assayed for ATP using the method of Beutler (Red cell metabolism. Amanual of biochemical methods. Third Edition, by Earnest Beutler, Grune& Stratton Inc., Orlando, Fla., 1984). Supernatant Hb was determined bythe tetramethylbenzidine method (Procedure No. 527, Sigma) (Standefer JC, Vanderagt D, Use of tetramethylbenzidine in plasma hemoglobin assay,Clin Chem, 1977;23:749-51). Total hemoglobin was determined by anautomated cell counter (Cell Dyn 3700, Abbott Laboratories, Abbott Park,Ill.). We measured extracellular potassium, pH, lactate and glucoseusing blood gas analyzers (RapidLab 348 or RapidLab 860, Bayer Corp).For morphology studies, we fixed RBCs in glutaraldehyde and scored 200cells by cell type: discocyte, 1.0; echinocyte I, 0.8; echinocyte II,0.6; echinocyte III, 0.4; echinospherocyte 0.2; and spherocyte, 0.0(Usry R T, Moore G L, Manalo F W, Morphology of stored, rejuvenatedhuman eiythrocytes, Vox Sang, 1975;28:176-83).

Statistics

Determination of means and standard deviation of experimental values,the performance of two-tailed, tests with Welch's correction, and linearregressions were carried out by using standard software (Instat,GraphPad Software, San Diego, Calif.). A value of p<0.05 was consideredsignificant. In a number of phototreated samples, no plaques, infectedcells or bacteria were visible in wells or culture dishes containingeither diluted or undiluted samples. In these circumstances, the extentof inactivation was calculated with the assumption that one plaque orcolony was observed, and the extent of pathogen inactivation wasreported as greater than the calculated value.

Results

RBC binding studies. A spectroscopic assay was developed to measure theeffect of DP on TP binding to RBCs suspended in Erythrosol. TP was addedto freshly prepared 20-percent hct RBCs to yield a final concentrationof 160 μM in suspensions containing or lacking 200 μM DP. Followingcentrifugation of RBCs, supernatant spectra were compared to spectra of160 μM TP added directly to supernatant. Results are given in FIG. 4.Addition of TP to RBC suspensions lacking DP results in a spectra(dashed line) whose 620 nm TP peak is reduced 70-percent from the peakof the spectra representing TP added directly to supernatant (solidline). The reduction of the TP peak when the dye is incubated with RBCssuggests that a substantial fraction of the dye is bound to cells.Addition of TP to RBCs containing DP results in supernatant spectra(dotted line) whose peak is partially restored to that of the dye addeddirectly to supernatant. The partial restoration of the supernatant TPpeak intensity in RBCs containing DP suggests that DP blocks TP bindingof some (approximately 36-percent), but not all sites in RBCs.

Virus inactivation studies. Virus inactivation experiments wereperformed in 20-percent hct RBCs suspended in Erythrosol and containing200 μM DP. The extent of inactivation using 1.1 J/cm² light was measuredas a function of TP dose. In general, the log₁₀ inactivation of eachvirus varied linearly with TP concentration (FIG. 5). Sensitivities toinactivation varied greatly among different viruses. Phototreatment ofRBC suspensions containing DP resulted in >8.4 log₁₀ of extracelluar VSVat 100 μM TP, >7.5 log₁₀ extracellular HIV at 80 μM TP, 6.2±0.1 log₁₀intracellular HIV at 80 μM TP, >6.3 log₁₀ extracellular PRV at 15 μMTP, >5.8 log₁₀ extracellular DHBV at 10 μM TP, and >6 log₁₀extracellular BVDV at 4 μM TP. With 100 μM TP, all tested viruses wereinactivated to the limit of assay detection. The vertical line in FIG. 5represents the 160 μM TP concentration used to assess RBC storageproperties following phototreatment.

Bacterial inactivation studies. Photoinactivation of gram positive andgram negative organisms was studied in experiments which utilized 100 μMTP and 200 μM DP in 20% hct RBCs suspended in Erythrosol. Results aregiven in Table 5. TP phototreatment resulted in >4 log₁₀ inactivation ofall tested organisms and >6 log₁₀ inactivation of 5 of 7 testedorganisms. TABLE 5 Bacterial inactivation by 100 μM TP, 200 μM DP and1.1 J/cm² red light Organism log₁₀ Inactivation S. epidermidis >7.6 S.aureus >6.3 Y. enterocolitica 5.9 ± 0.6 E. coli 7.1 ± 0.5 P. fluorescens5.4 ± 1.0 S. liquifaciens >7.1 S. marcescens 6.2 ± 1.0n = 4

RBC storage studies. FIG. 6 shows the effect of dipyridamole and choiceof additive solution on hemolysis following the storage of phototreatedRBCs (160 μM TP and 1.1 J/cm² light). In the presence of 200 μM DP, TPphototreated RBC suspended in Erythrosol (panel A) had low levels(0.29%) of hemolysis following 42 day 1-6° C. storage, but were elevatedcompared to controls (0.19%) (p<0.05, n=10). Much less hemolysis wasobserved in phototreated RBCs suspended in Erythrosol and lacking DPthan those stored in ADSOL and lacking DP (0.46±0.1 vs. 24.56±7.57 atday 42). Although DP significantly (p<0.05) reduced photoinducedhemolysis of RBCs suspended in both additive solutions, its effect wasmuch more evident in cells suspended in ADSOL than those suspended inEzythrosol.

The effect of 160 μM TP, 200 μM DP and 1.1 J/cm² light on morphologyscore, pH, glucose utilization, lactate production, and ATP levels ofRBCs suspended in Erytrosol is given in FIG. 7, panels A through E,respectively. TP phototreatment resulted in acceptable but significantly(p<0.05) different morphology scores (94.5±1.5 vs. 98.3±0.7, n=4) andextracellular pHs (6.48±0.07 vs. 6.38±0.06, n=10) at 42 days of storage.Although glucose levels were greater in phototreated samples because oftheir dilution (to 20-percent hct) and resuspension with Erytlrosol (to45-percent hct), similar (p>0.05) glucose utilization rates (6.20±0.97vs. 7.08±0.31 mM/day, n=4), and lactose production (26.7±1.9 vs.28.2±1.0 mM on day 42, n=4) were observed in treated and controlsamples. ATP levels of phototreated samples declined with storage morerapidly than controls (2.63±0.07 vs. 3.61±0.22 μmole/g Hb, day 42,p<0.05, n=4).

FIG. 8 shows the effect of DP on potassium release from RBCs suspendedin Erythrosol and treated with 160 μM TP and 1.1 J/cm² light. Potassiumleakage from phototreated RBCs is very rapid, with 38.4±4.4 mM potassiumreleased to the supernatant after one day of storage compared to 4.4±0.4mM in controls (p<0.05, n=10). In the presence of DP, potassium effluxfrom phototreated RBCs is partially inhibited, with 16.6±4.2 mM in thesupernatant after one day of storage (p<0.05 compared to either controlor phototreated samples lacking DP, n=10). Very similar results wereobtained in control and phototreated RBCs suspended in ADSOL (data notshown).

1. A method for decontaminating a biological fluid comprising: (a)contacting a biological fluid with a virucidal effective amount of adiphenylpyrilium dye; and (b) irradiating the resulting mixture withlight of about 560 to about 800 nm to achieve a virucidal effect.
 2. Themethod of claim 1, wherein the biological fluid is selected from thegroup consisting of: blood, cellular blood components, liquid bloodcomponents; and mixtures of cellular and liquid blood components.
 3. Themethod of claim 1, wherein the diphenylpyrilium dye is a compound of theformula:

wherein: Y is S or Se; R₁ and R₂ are independently selected from thegroup consisting of amino, allylamino, and alkoxyamino; and R₃ is alkyl,alkoxy, or aryl, arylamino, and arylalkoxyamino.
 4. The method of claim1 wherein the virucidal effective amount is that sufficient to achieve avirus kill capacity of at least about 6.0 log₁₀ extracellular virus inthe biological fluid.
 5. The method of claim 1 wherein the virucidaleffective amount is that sufficient to achieve a virus kill capacity ofat least about 3.5 log₁₀ intracellular virus in the biological fluid. 6.The method of claim 1 wherein the step of contacting a biological fluidwith a virucidal effective amount of a diphenylpyrilium dye furthercomprises the addition of dipyridamole.
 7. The method of claim 5 whereinthe decontamination causes less than about 3% hemolysis in a red bloodcell containing biological fluid upon exposure to the diphenylpyriliumdye for about 42 days at about 1-6° C.
 8. A method for decontaminating abiological fluid comprising: (a) adding to the biological fluiddipyridamole and a virucidal effective amount of a diphenylpyrilium dyeof the formula:

wherein Y is S or Se; R₁ and R₂ are independently amino, methylamino, ordimethylamino; and R₃ is alkyl of 1-6 carbons or phenyl; and (b)irradiating the resulting mixture with at least about 0.025 J/cm² oflight of about 560 nm to about 800 nm to decontaminate said biologicalfluid.
 9. The method of claim 8 wherein the dipyridamole is added to thebiological fluid to a concentration of at least about 100 μM.
 10. Themethod of claim 8 wherein the irradiation step comprises irradiatingsaid biological fluid with about 0.1 to about 1.0 J/cm² of light ofabout 590 nm to about 640 nm wavelength.
 11. The method of claim 8wherein Y is S; R₁ and R₂ are both dimethylamino; and R₃ is methyl. 12.The method of claim 11 wherein the virucidal effective amount is thatsufficient to achieve at least about a 30 μM diphenylpyriliumconcentration in the biological fluid.
 13. The method of claim 11wherein the virucidal effective amount is that sufficient to achieve atleast about a 160 μM diphenylpyrilium concentration in the biologicalfluid.
 14. The method of claim 8 wherein the virucidal effective amountconstitutes sufficient diphenylpyrilium to achieve a virus kill capacitycorresponding to at least about 7.0 log₁₀ extracellular VSV in thebiological fluid.
 15. The method of claim 8 wherein the virucidaleffective amount constitutes sufficient diphenylpyrilium to achieve avirus kill capacity corresponding to at least about 3.5 log₁₀intracellular VSV in the biological fluid.
 16. The method of claim 8wherein the biological fluid is selected from the group consisting of:cellular blood components, liquid blood components; and mixtures ofcellular and liquid blood components.
 17. The method of claim 8 whereinthe decontamination inactivates gram positive and gram negativeorganisms to a level of greater than 6 log₁₀.
 18. The method of claim 8wherein the decontaminated fluid experiences less than about 1%hemolysis over a period of about 42 days following decontamination atabout 1-6° C.
 19. A method for decontaminating a biological fluidcomprising: (a) adding 2′,4′-bis(4-N,N-dimethylaminophenyl)6′methylthiopyrilium iodide to the biological fluid to a concentrationof about 50 to about 300 μM; (b) adding dipyridamole to the biologicalfluid to a concentration of about 100 to about 300 μM; and (c)irradiating the resulting biological fluid with light of about 560 toabout 800 nm.